Our object was to determine how innervation regulates muscle insulin sensitivity. Insulin-stimulated uptake of the nonmetabolized amino acid, 2-amino-isobutyric acid, was used as a measure of insulin sensitivity in denervated rat extensor digitorum longus muscles retaining either a similar 2.5-cm (""proximal denervation'') or a similar to 0.5-cm (""distal denervation'') length of distal nerve stump. Because both muscles were inactive in the first 24 h, any difference in insulin sensitivity could be due only to some trophic influence of the distal nerve stump. Fifteen hours after either type of denervation, 2-aminoisobutyric acid uptake was refractory to insulin. However, at 24 h, insulin sensitivity of distally denervated muscles (with or without a second ipsilateral proximal denervation) was normal, whereas that of proximally denervated muscles was still relatively insensitive. In the absence of insulin, the two types of denervated muscles at 24 h showed no difference in 2-aminoisobutyric acid uptake. Finally, organ culture of paired muscles with or without long nerve stumps showed corresponding differences in insulin-stimulated 2-aminoisobutyric acid uptake after 48 h in vitro. Thus, a neurotrophic factor, independent of impulse activity, stretch, or changes in blood flow, regulates one type of muscle insulin sensitivity.